NO319148B1 - Sulfide oxidizing bacteria and process with them - Google Patents
Sulfide oxidizing bacteria and process with them Download PDFInfo
- Publication number
- NO319148B1 NO319148B1 NO19962861A NO962861A NO319148B1 NO 319148 B1 NO319148 B1 NO 319148B1 NO 19962861 A NO19962861 A NO 19962861A NO 962861 A NO962861 A NO 962861A NO 319148 B1 NO319148 B1 NO 319148B1
- Authority
- NO
- Norway
- Prior art keywords
- campylobacter
- sulfide
- fluid
- nrrl
- cvo
- Prior art date
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- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 27
- 230000001590 oxidative effect Effects 0.000 title claims abstract description 13
- 230000008569 process Effects 0.000 title abstract description 4
- 241000894006 Bacteria Species 0.000 title description 31
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- -1 sulfide compound Chemical class 0.000 claims abstract description 14
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- 241000589994 Campylobacter sp. Species 0.000 claims description 14
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical group S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 claims description 10
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
- B01D53/84—Biological processes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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Abstract
En bakteriekultur som omfatter en Campylobacterlignende art er tilveiebrakt. Bakteriekulturen er i stand til å oksidere en sulfidforbindelse i en fluid så som f.eks. en produsert saltoppløsning. Videre er det tilveiebrakt en fremgangsmåte for hovedsakelig å redusere sulfidinnholdet i en fluid hvori prosessen omfatter å bringe fluidet i berøring med en bakteriekultur som omfatter en Campylobacter-lignende art.A bacterial culture comprising a Campylobacter-like species is provided. The bacterial culture is capable of oxidizing a sulfide compound in a fluid such as e.g. a produced saline solution. Furthermore, a method is provided for substantially reducing the sulfide content of a fluid in which the process comprises contacting the fluid with a bacterial culture comprising a Campylobacter-like species.
Description
Foreliggende oppfinnelse angår bakterier som er istand til å oksidere en sulfidforbindelse eller hovedsakelig redusere sulfidinnholdet i saltoppløsninger, olje, gass eller kombinasjon av en eller flere derav. Foreliggende oppfinnelse angår også en fremgangsmåte for å redusere sulfidinnholdet i saltoppløsninger, olje, gass eller kombinasjoner av to eller flere derav. The present invention relates to bacteria which are able to oxidize a sulphide compound or mainly reduce the sulphide content in salt solutions, oil, gas or a combination of one or more of these. The present invention also relates to a method for reducing the sulphide content in salt solutions, oil, gas or combinations of two or more thereof.
Sulfider, spesielt oppløselige sulfider (H2S, HS", S<2>" eller kombinasjoner derav), blir ofte oppdaget i saltoppløsninger såsom f.eks. oljefeltsaltoppløsninger som en konsekvens av aktiviteten til sulfatreduserende bakterier (SRB), og gir alvorlige problemer for industrien pga. deres toksisitet, lukt, korroderende natur og potensiale for å plugge brønnhullet. Foreliggende behandlingsteknologier for å fjerne sulfid inkluderer fysikalsk/kjemiske fremgangsmåter såsom stripping med damp eller røykgass, luftoksidasjon og utfelling. Mikrobiell behandling kan imidlertid være et mer effektivt og kostnadseffektivt alternativ for å redusere sulfidnivået. Sulfides, especially soluble sulfides (H2S, HS", S<2>" or combinations thereof), are often detected in salt solutions such as e.g. oilfield salt solutions as a consequence of the activity of sulphate-reducing bacteria (SRB), and cause serious problems for the industry due to their toxicity, odor, corrosive nature and potential to plug the wellbore. Existing treatment technologies to remove sulphide include physical/chemical methods such as steam or flue gas stripping, air oxidation and precipitation. However, microbial treatment may be a more effective and cost-effective option for reducing sulphide levels.
Mineraloljereservoarer har adskilte mikrobielle kolonier som inneholder en variasjon av fysiologiske bakterietyper. Gjærende bakterier, hydrokarbon-oksiderende, denitrifikerende, metanogene og SRB har alle blitt isolert fra reservoarsaltoppløsninger. SRB er av stor betydning for petroleumsindustrien pga. deres evne til å redusere sulfater til sulfider for derved å bidra på en ødeleggende måte til å plugge injeksjonsbrønner, korrosjon av utstyr og surgjøring av gass, olje eller begge. Omkostningene for oljeproduksjonen økes betydelig pga. utstyrssvikt, tilleggsutstyr som er nødvendig for å fjerne sulfid, behov for biocider.til å kontrollere mikrobiell vekst og ytterligere kjemikalier nødvendig for å fjerne eller hindre jernsulfidskall. Mineral oil reservoirs have distinct microbial colonies containing a variety of physiological bacterial types. Fermenting, hydrocarbon-oxidizing, denitrifying, methanogenic, and SRB bacteria have all been isolated from reservoir brines. SRB is of great importance to the petroleum industry because their ability to reduce sulfates to sulfides thereby contributing in a destructive way to plugging injection wells, corrosion of equipment and acidification of gas, oil or both. The costs for oil production are increased significantly due to equipment failure, additional equipment required to remove sulphide, need for biocides to control microbial growth and additional chemicals required to remove or prevent iron sulphide scaling.
Sulfidproduksjonen avhenger generelt av et antall ernærings- og fysikalske faktorer som påvirker veksten av SRB i f.eks. oljereservoarer. Konsentrasjonene av utnyttbart karbon, sulfat, nitrogen og fosfor påvirker også veksten av SRB og sulfatreduksjonshastigheter. Sulfide production generally depends on a number of nutritional and physical factors that affect the growth of SRB in e.g. oil reservoirs. The concentrations of usable carbon, sulfate, nitrogen, and phosphorus also affect the growth of SRB and sulfate reduction rates.
Andre bakterier kan også spille en rolle ved korrosjon og reservoarsurgjøring. F.eks. har tallrike stammer av Shewenella putreficians blitt isolert fra oljefeltsaltoppløsninger og relaterte væsker som kan vokse anaerobt ved å redusere svoveloksyanioner andre enn sulfat til hydrogensulfid. Other bacteria can also play a role in corrosion and reservoir acidification. E.g. numerous strains of Shewenella putreficians have been isolated from oil field brines and related fluids that can grow anaerobically by reducing sulfur anions other than sulfate to hydrogen sulfide.
Tradisjonelt har oljeindustrien benyttet biocider, såsom f.eks. kvaternære ammoniumforbindelser, isotiazolonderivater, glutaraldehyd, formaldehyd, akrolein eller kombinasjonen av hvilken som helst to eller flere av disse til å kontrollere SRB. Suksessen med denne tilnærming er imidlertid begrenset pga. bakterienes tilbøyelighet til å danne biofilmer som er relativt impermeable for biocider. Biologiske tilnærminger for kontroll av SRB er blitt undersøkt som alternativer til fysikalsk/kjemisk behandling. Tilsetting av høye konsentrasjoner av nitrat til anrikingskulturer forbedret med sulfat og forskjellige elektrondonorer, er blitt rapportert å resultere i inhibisjon av biogensulfidproduksjon i lange tidsavsnitt. Traditionally, the oil industry has used biocides, such as e.g. quaternary ammonium compounds, isothiazolone derivatives, glutaraldehyde, formaldehyde, acrolein or the combination of any two or more of these to control SRB. However, the success of this approach is limited due to the bacteria's tendency to form biofilms that are relatively impermeable to biocides. Biological approaches to control SRB have been investigated as alternatives to physical/chemical treatment. Addition of high concentrations of nitrate to enrichment cultures enhanced with sulfate and various electron donors has been reported to result in inhibition of biogenic sulfide production for long periods of time.
Nitrat har også blitt brukt som en elektronakseptor for anaerob sulfidoksidering. Nitratavhengig sulfidoksidering ved endogene bakterier i vann assosiert med produksjon av olje, gass eller begge er blitt demonstrert i laboratoriestudier med stenkjerner såvel som i felttester, hvor sulfidnivåene falt 40 til 60% i saltoppløsning fra tre tilstøtende produksjonsbrønner 45 dager etter injeksjon av nitrat inn i formasjonen. Mesteparten av forskningen på biooksidasjon av sulfid i saltoppløsninger, gasstrømmer og råolje har fokusert på anvendelse av eksogene arter av Thiobacillus. I en feltdemonstrasjon for å helbrede surt produsert vann, oksiderte Thiobacillus denitrificans, stamme F, effektivt sulfid aerobt til sulfat, på tross av flere mangler ved systemet. Nitrate has also been used as an electron acceptor for anaerobic sulfide oxidation. Nitrate-dependent sulfide oxidation by endogenous bacteria in water associated with production of oil, gas, or both has been demonstrated in laboratory rock core studies as well as in field tests, where sulfide levels dropped 40 to 60% in brine from three adjacent production wells 45 days after injection of nitrate into the formation . Most of the research on the biooxidation of sulphide in brines, gas streams and crude oil has focused on the use of exogenous species of Thiobacillus. In a field demonstration to heal acidic produced water, Thiobacillus denitrificans, strain F, efficiently aerobically oxidized sulfide to sulfate, despite several deficiencies of the system.
Å oksidere sulfider til sulfater synes ikke å være løsningen fordi sulfater kan igjen bli redusert av SRB til sulfider og derved skape problemene illustrert ovenfor. Derfor er det et stadig økende behov for å utvikle en bakteriekultur som kan oksidere et sulfid eller deler derav til elementært svovel og å utvikle en fremgangsmåte for hovedsakelig å oksidere et sulfid, eller hovedsakelig redusere sulfidinnholdet i en fluid såsom saltoppløsning, olje, gass eller kombinasjoner av to eller flere av disse. Utvikling av slike bakterielle kulturer eller fremgangsmåter eller begge vil også i stor grad bidra til bedre forståelse av anvendelser, begrensninger eller kombinasjoner derav i biobehandlinger av saltoppløsninger, olje, gass eller kombinasjoner av to eller flere av disse. Oxidizing sulfides to sulfates does not seem to be the solution because sulfates can again be reduced by SRB to sulfides and thereby create the problems illustrated above. Therefore, there is an ever-increasing need to develop a bacterial culture that can oxidize a sulfide or parts thereof to elemental sulfur and to develop a method to mainly oxidize a sulfide, or mainly reduce the sulfide content in a fluid such as brine, oil, gas or combinations of two or more of these. Development of such bacterial cultures or methods or both will also largely contribute to a better understanding of applications, limitations or combinations thereof in biotreatments of salt solutions, oil, gas or combinations of two or more of these.
EP 0 218 958 A2 og US patent nr. 4,760,027 beskriver en fremgangsmåte til desulfurisering av gasser ved mikrobiologiske teknikker ved å bruke Thiobacillus denitrificans til å oksidere hydrogensulfid til sulfatforbindelser. WO 95/24960 beskriver en mikroorganisme som tilhører slekten Thiobacillus som degraderer hydrogensulfid til sulfat. US patent 5,366,891 beskriver anvendelse av mikroorganismer av slekten Thiobacillus til å konvertere metallsulfider til metallsulfat. US patent 5,236,677 angår en fremgangsmåte til å fjerne illeluktende svovelforbindelser, for eksempel hydrogensulfid, ved å bruke mikroorganismer i Thiobacillus familien. US 5,196,129 angår stabile, enkeltfasede oppløsninger av vann-i-olje mikroemulsjoner som inneholder mikroorganismer. US patent 4,968,622 angår degradering av svovelholdige forurensinger, for eksempel hydrogensulfid til sulfat. Brock, Madigan, Martinko, Parker: Biology of Microorganisms, syvende utgave, Prentice-Hall, 1994, ISBN 0-13-176660-0 beskriver flere bakterier, for eksempel svovelbakterier og forskjellige sykler, for eksempel redox sykler for nitrogen og svovel. EP 0 218 958 A2 and US patent no. 4,760,027 describe a method for desulphurisation of gases by microbiological techniques using Thiobacillus denitrificans to oxidize hydrogen sulphide to sulphate compounds. WO 95/24960 describes a microorganism belonging to the genus Thiobacillus which degrades hydrogen sulphide to sulphate. US patent 5,366,891 describes the use of microorganisms of the genus Thiobacillus to convert metal sulfides to metal sulfate. US patent 5,236,677 relates to a method for removing malodorous sulfur compounds, for example hydrogen sulphide, by using microorganisms in the Thiobacillus family. US 5,196,129 relates to stable, single-phase solutions of water-in-oil microemulsions containing microorganisms. US patent 4,968,622 relates to the degradation of sulphur-containing pollutants, for example hydrogen sulphide to sulphate. Brock, Madigan, Martinko, Parker: Biology of Microorganisms, seventh edition, Prentice-Hall, 1994, ISBN 0-13-176660-0 describes several bacteria, such as sulfur bacteria, and different cycles, such as redox cycles for nitrogen and sulfur.
En hensikt med foreliggende oppfinnelse er derfor å tilveiebringe en bakteriell kultur eller en bakterie som er istand til hovedsakelig å oksidere et sulfid, eller hovedsakelig redusere sulfidinnholdet i en fluid såsom saltoppløsning, olje, gass eller kombinasjoner av to eller flere av disse. En annen hensikt med foreliggende. oppfinnelse er å tilveiebringe en fremgangsmåte til hovedsakelig å oksidere et sulfid eller hovedsakelig å redusere sulfidinnholdet i en fluid såsom saltoppløsning, olje, gass eller kombinasjoner av to eller flere av disse. Andre hensikter og trekk vil bli klart når oppfinnelsen blir mer fullstendig beskrevet nedenfor. One purpose of the present invention is therefore to provide a bacterial culture or a bacterium which is capable of mainly oxidizing a sulphide, or mainly reducing the sulphide content in a fluid such as salt solution, oil, gas or combinations of two or more of these. Another purpose of the present. invention is to provide a method to mainly oxidize a sulphide or mainly to reduce the sulphide content in a fluid such as salt solution, oil, gas or combinations of two or more of these. Other purposes and features will become clear when the invention is more fully described below.
Disse hensikter er oppnådd med foreliggende oppfinnelse kjennetegnet ved det som fremgår av de vedlagte krav. These purposes have been achieved with the present invention characterized by what appears from the attached claims.
I henhold til den første utforming av foreliggende oppfinnelse er det tilveiebragt en bakteriekultur som er istand til hovedsakelig å oksidere sulfid eller hovedsakelig redusere sulfidinnholdet i en fluid. According to the first embodiment of the present invention, a bacterial culture is provided which is capable of mainly oxidizing sulphide or mainly reducing the sulphide content in a fluid.
I henhold til en annen utforming av foreliggende oppfinnelse er det tilveiebragt en prosess for hovedsakelig å oksidere et sulfid eller hovedsakelig redusere sulfidinnholdet i en fluid som omfatter å bringe fluidet i kontakt med en blanding som omfatter en bakteriekultur som er istand til å oksidere sulfid i en fluid. According to another embodiment of the present invention, there is provided a process for mainly oxidizing a sulphide or mainly reducing the sulphide content in a fluid which comprises bringing the fluid into contact with a mixture comprising a bacterial culture capable of oxidising sulphide in a fluid.
Fig. 1 illustrerer sulfidoksidasjon ved Campylobacter sp. CVO (NRRL B-21472) i en filtrert saltoppløsning som inneholder eksogent tilsatt kaliumnitrat og natriumfosfat (monobasis). Fig. 2 viser sulfidoksidasjon ved Campylobacter sp. CVO (NRRL B-21472) i CSB/DTA medium. Fig. 3 illustrerer den synergistiske effekt som oppstår når Campylobacter sp. CVO (NRRL B-21472) kombineres med natriumnitrat og natriumfosfat (monobasis) i en produsert saltoppløsning ved sulfidoksidasjon. Fig. 1 illustrates sulphide oxidation by Campylobacter sp. CVO (NRRL B-21472) in a filtered saline solution containing exogenously added potassium nitrate and sodium phosphate (monobase). Fig. 2 shows sulphide oxidation by Campylobacter sp. CVO (NRRL B-21472) in CSB/DTA medium. Fig. 3 illustrates the synergistic effect that occurs when Campylobacter sp. CVO (NRRL B-21472) is combined with sodium nitrate and sodium phosphate (monobase) in a produced salt solution by sulfide oxidation.
Betegnelsen "sulfid" anvendt i denne oppfinnelsen er generisk referert til som, med mindre noe annet er angitt, uorganiske sulfider, organiske sulfider eller kombinasjoner av to eller flere derav som inneholder en gjentagende enhet av -S„- i sulfidmolekylet, hvor n er et tall fra 1 til 10, fortrinnsvis 1 til ca. 5, og mest fortrinnsvis 1 til 3. Sulfidforbindelsen kan være oppløselig, uoppløselig, hovedsakelig oppløselig eller hovedsakelig uoppløselig i vandig media, ikke-vandige media eller kombinasjoner derav. Oppløselige sulfider som beskrevet ovenfor kan være H2S, HS", S<2>" eller kombinasjoner av to eller flere av disse. The term "sulfide" used in this invention generically refers to, unless otherwise indicated, inorganic sulfides, organic sulfides, or combinations of two or more thereof containing a repeating unit of -S„- in the sulfide molecule, where n is a numbers from 1 to 10, preferably 1 to approx. 5, and most preferably 1 to 3. The sulphide compound may be soluble, insoluble, mainly soluble or mainly insoluble in aqueous media, non-aqueous media or combinations thereof. Soluble sulfides as described above can be H2S, HS", S<2>" or combinations of two or more of these.
Eksempler på sulfid forbindelser som kan bli hovedsakelig oksidert eller fjernet inkluderer, men er ikke begrenset til, hydrogensulfid, dimetylsulfid, dimetyldisulfid, dietylsulfid, dietyldisulfid, natriurrisulfid, narriumhydrosulfid, kaliumhydrosulfid, kaliumsulfid, jernsulfid og alle kombinasjoner av hvilken som helst to eller flere derav. Examples of sulfide compounds that may be substantially oxidized or removed include, but are not limited to, hydrogen sulfide, dimethyl sulfide, dimethyl disulfide, diethyl sulfide, diethyl disulfide, sodium uric sulfide, sodium hydrosulfide, potassium hydrosulfide, potassium sulfide, iron sulfide, and any combination of any two or more thereof.
I henhold til foreliggende oppfinnelse betegner "fluid" en væske, en gass eller kombinasjoner derav. Eksempler på fluider som er egnet til anvendelse i foreliggende oppfinnelse inkluderer, men er ikke begrenset til, saltoppløsninger, olje, gass eller kombinasjoner to eller flere derav. Betegnelsen "saltoppløsning" eller "saltoppløsninger" brukt i denne oppfinnelse er referert til som, med mindre noe annet er angitt, vann, en oppløsning, en suspensjon eller kombinasjoner av to eller flere derav. Generelt inneholder en oppløsning oppløselige stoffer såsom salter. Suspensjonen kan også inneholde oppløste, delvis oppløste eller uoppløste stoffer såsom salter. Eksempler på salter inkluderer, men er ikke begrenset til, metallsalter, såsom f.eks. natriumklorid, kaliumklorid, kalsiumklorid, kalsiumbromid, magnesiumklorid, magnesiumbromid, natriumdikarbonat, natriumsulfat, ammoniumklorid, natriumsulfid, natriumhydrosulfid, kaliumhydrosulfid, kaliumsulfid, jernsulfid og kombinasjoner av to eller flere derav. Generelt kan det totale saltinnholdet i en oppløsning eller suspensjon variere i stor grad fra f.eks. ca. 0,5 til så høyt som ca. 50 vekt%. Den for tiden foretrukkede saltoppløsning er en produsert saltoppløsning som noen ganger også refereres til som oljefeltsaltoppløsning, eller produsert vann, eller mineraloljesaltoppløsning eller reservoarsaltoppløsning og er en saltoppløsning produsert sammen med olje, gass eller begge to. En produsert saltoppløsning er generelt kontaminert med noe olje, og/eller gass. According to the present invention, "fluid" denotes a liquid, a gas or combinations thereof. Examples of fluids suitable for use in the present invention include, but are not limited to, salt solutions, oil, gas or combinations of two or more thereof. The term "salt solution" or "salt solutions" used in this invention refers to, unless otherwise indicated, water, a solution, a suspension, or combinations of two or more thereof. In general, a solution contains soluble substances such as salts. The suspension may also contain dissolved, partially dissolved or undissolved substances such as salts. Examples of salts include, but are not limited to, metal salts, such as e.g. sodium chloride, potassium chloride, calcium chloride, calcium bromide, magnesium chloride, magnesium bromide, sodium dicarbonate, sodium sulfate, ammonium chloride, sodium sulfide, sodium hydrosulfide, potassium hydrosulfide, potassium sulfide, iron sulfide and combinations of two or more thereof. In general, the total salt content of a solution or suspension can vary greatly from e.g. about. 0.5 to as high as approx. 50% by weight. The presently preferred brine is a produced brine which is sometimes also referred to as oil field brine, or produced water, or mineral oil brine or reservoir brine and is a brine produced together with oil, gas or both. A produced salt solution is generally contaminated with some oil and/or gas.
I henhold til en første utforming av foreliggende oppfinnelse oppfatter en bakteriekultur, eller består essensielt av, eller består av sulfidoksiderende bakterier som er tilveiebragt som er istand til å oksidere sulfidforbindelse i en sulfidholdig fluid. Oksidasjonsproduktet av sulfid eller deler derav i foreliggende oppfinnelse omfatter generelt elementært svovel. Betegnelsen "deler" er brukt heri for å angir enhver brøkdel av sulfid. Bakterien er Campylobacter-lignende arter. According to a first embodiment of the present invention, a bacterial culture perceives, or essentially consists of, or consists of, sulfide-oxidizing bacteria that are provided and are capable of oxidizing sulfide compounds in a sulfide-containing fluid. The oxidation product of sulphide or parts thereof in the present invention generally comprises elemental sulphur. The term "parts" is used herein to denote any fractional part of sulfide. The bacteria are Campylobacter-like species.
Bakterier som er kjent for å oksidere en sulfidforbindelse produserer generelt en sulfatforbindelse. Slike bakterier, f.eks. Thiobacilli, oksiderer generelt ikke en sulfidforbindelse til elementært svovel. Bakteriene beskrevet i denne søknad oksiderer imidlertid en sulfidforbindelse eller deler derav til dementert svovel, spesielt i blandede kulturer og eliminerer derved problemet med produksjon av sulfat som i sin tur reduseres av SRB til en sulfidforbindelse. Oksidering av sulfid til elementært svovel er virkelig overraskende. Bacteria known to oxidize a sulfide compound generally produce a sulfate compound. Such bacteria, e.g. Thiobacilli, generally do not oxidize a sulfide compound to elemental sulfur. However, the bacteria described in this application oxidize a sulphide compound or parts thereof to demented sulphur, especially in mixed cultures and thereby eliminate the problem of production of sulphate which in turn is reduced by SRB to a sulphide compound. Oxidation of sulfide to elemental sulfur is truly surprising.
Disse nye bakteriene ble isolert ved anriking av en produsert saltoppløsning oppnådd fra saltoppløsninger oppsamlet fra fritt vanns væskeutskillingstank i Coleville Unit, Coleville, Saskatchewan, Canada. Anrikingen ga to stammer av bakterier som er blitt gitt laboratoriebetegnelsene CVO og FWKO B, og gitt aksesjonsnummerene NRRL B-21472 og NRRL B-21473. These new bacteria were isolated by enrichment of a manufactured saline solution obtained from brines collected from the free water liquid separation tank at the Coleville Unit, Coleville, Saskatchewan, Canada. The enrichment yielded two strains of bacteria which have been given the laboratory designations CVO and FWKO B, and given the accession numbers NRRL B-21472 and NRRL B-21473.
Designeringene NRRL B-21472 og NRRL B-21473 reflekterer det faktum at bakteriekulturene CVO og FWKO B er blitt deponert ved en offentlig deponeringsinstitusjon, United States Department of Agriculture, Agricultural Research Service, Northern Regional Research Laboratory, Peoria, Illinois 61604, USA. Deponeringen er blitt foretatt under Budapestavtalen og i henhold til praksis i United States Patent and Trademark Office slik at alle restriksjoner på tilgjengelighet av stammene til offentligheten vil ugjenkallelig fjernes ved at det gis patent på denne søknad i hvilken disse viktige, nye stammer er gjenstanden. Således vil stammene være tilgjengelig for offentligheten for utnyttelse i henhold til foreliggende oppfinnelse. The designations NRRL B-21472 and NRRL B-21473 reflect the fact that the bacterial cultures CVO and FWKO B have been deposited at a public depository, United States Department of Agriculture, Agricultural Research Service, Northern Regional Research Laboratory, Peoria, Illinois 61604, USA. The deposit has been made under the Budapest Agreement and in accordance with the practice of the United States Patent and Trademark Office so that all restrictions on the availability of the strains to the public will be irrevocably removed by the grant of a patent on this application in which these important new strains are the subject. Thus, the strains will be available to the public for utilization according to the present invention.
Fremsatt i tabell I nedenfor er konsentrasjonen av forskjellige elementer brukt i anriket medium for å dyrke de nye stammene til Campylobacter sp.-stammer NRRL B-21472 og NRRL B-21473. Konsentrasjonen er i hvert eksempel uttrykt av elementet, skjønt det er anerkjent at alt eller del av hver kan være tilstede i form av et oppløselig ion, således hvor P er tilstede i en kombinert form, f.eks. som fosfat. Svovel er ønskelig anvendt i form av sulfat. Noen av de nødvendige metaller er fordelaktig tilsatt i form av et sulfat. Således er minimumkonsentrasjonene for svovel normalt overskredet. Fortrinnsvis er magnesium, kalsium, jern, zink, kobber, mangan og kobolt anvendt i form av et sulfat eller i form av en forbindelse som er konvertert in situ til et sulfat. Fortrinnsvis er molybden og bor anvendt i en oppløselig form såsom f.eks. henholdsvis molybdat og borat. Kalium er fortrinnsvis anvendt som et sulfat eller fosfat, eller i form av en forbindelse som er konvertert in situ til et sulfat eller fosfat. Fosfor er fortrinnsvis anvendt i form av fosforsyre eller i form av et fosfat (monobasis), eller fosfat (dibasis), f.eks. som et kalium- eller ammoniumsalt eller som en forbindelse som er konvertert in situ til et slikt salt. Mens nitrogen også er nødvendig for produksjonen av cellemasse, er det ikke fremsatt noe minimumsnivå ovenfor pga. at slike minimumsverdier lett kan bli avhengig av ønsket cellemasse og fordi en nitrogenholdig forbindelse er brukt som midler til å dyrke cellemasse. Set forth in Table I below are the concentrations of various elements used in enriched medium to grow the new strains of Campylobacter sp. strains NRRL B-21472 and NRRL B-21473. The concentration is in each example expressed by the element, although it is recognized that all or part of each may be present in the form of a soluble ion, thus where P is present in a combined form, e.g. as phosphate. Sulfur is preferably used in the form of sulphate. Some of the necessary metals are advantageously added in the form of a sulphate. Thus, the minimum concentrations for sulfur are normally exceeded. Magnesium, calcium, iron, zinc, copper, manganese and cobalt are preferably used in the form of a sulphate or in the form of a compound which is converted in situ to a sulphate. Preferably, molybdenum and boron are used in a soluble form such as e.g. molybdate and borate respectively. Potassium is preferably used as a sulphate or phosphate, or in the form of a compound which is converted in situ to a sulphate or phosphate. Phosphorus is preferably used in the form of phosphoric acid or in the form of a phosphate (monobase), or phosphate (dibase), e.g. as a potassium or ammonium salt or as a compound converted in situ to such a salt. While nitrogen is also necessary for the production of cell mass, no minimum level has been set out above due to that such minimum values can easily become dependent on the desired cell mass and because a nitrogenous compound is used as means to grow cell mass.
Generelt kan enhver uorganisk eller organisk nitrogenholdig forbindelse anvendes som en nitrogenkilde. Den for tiden foretrakkede nitrogenkilde er en uorganisk nitrogenholdig forbindelse såsom f.eks. ammoniumsalter, metallnitratsalter eller kombinasjoner av hvilken som helst to eller flere derav. Eksempler på egnede nitrogenkilder inkluderer, men er ikke begrenset til, ammoniakk, ammoniumnitrat, ammoniumklorid, ammoniumsulfat, natriumnitrat, kaliumnitrat, magnesiumnitrat og kombinasjoner av hvilke som helst to eller flere derav. Enhver organisk forbindelse som er generelt anvendt for å understøtte vekst av mikroorganismer kan anvendes som kilder for karbon eller energi eller begge. Den for tiden foretrakkede karbon- eller energikilde er et acetat. Andre elementer såsom natrium, selen, jod, kan også være tilstede i vekstmediet. In general, any inorganic or organic nitrogen-containing compound can be used as a nitrogen source. The currently preferred nitrogen source is an inorganic nitrogenous compound such as e.g. ammonium salts, metal nitrate salts or combinations of any two or more thereof. Examples of suitable nitrogen sources include, but are not limited to, ammonia, ammonium nitrate, ammonium chloride, ammonium sulfate, sodium nitrate, potassium nitrate, magnesium nitrate, and combinations of any two or more thereof. Any organic compound that is generally used to support the growth of microorganisms can be used as sources of carbon or energy or both. The currently preferred carbon or energy source is an acetate. Other elements such as sodium, selenium, iodine, may also be present in the growth medium.
Bakteriene i henhold til foreliggende oppfinnelse kan dyrkes i enhver egnet beholder i fravær av oksygen. Veksttémperaturen kan variere noe, men generelt i området fra ca. 10°C til ca. 40°C, fortrinnsvis ca. 10°C til ca. 35°C og mest fortrinnsvis 20°C til 35°C. Bakteriene kan dyrkes under et variasjon av trykk i området fra ca. 0,5 til ca. 15 atmosfærer (atm), fortrinnsvis ca. 0,5 til ca. 10 atmosfærer og mest fortrinnsvis 0,9 til 5 atmosfærer. pH-verdien til vekstmediet kan variere fra 5 til ca. 8,5, fortrinnsvis fra ca. 6 til ca. 8,5 og mest fortrinnsvis 7 til 8. The bacteria according to the present invention can be cultivated in any suitable container in the absence of oxygen. The growth temperature can vary somewhat, but generally in the area from approx. 10°C to approx. 40°C, preferably approx. 10°C to approx. 35°C and most preferably 20°C to 35°C. The bacteria can be cultivated under a variation of pressure in the area from approx. 0.5 to approx. 15 atmospheres (atm), preferably approx. 0.5 to approx. 10 atmospheres and most preferably 0.9 to 5 atmospheres. The pH value of the growth medium can vary from 5 to approx. 8.5, preferably from approx. 6 to approx. 8.5 and most preferably 7 to 8.
Fremgangsmåten med hensyn til oppfinnelsen kan utføres kontinuerlig. F.eks. det å bringe en fluid i kontakt med den bakterielle kultur kan gjøres ved å anvende kontinuerlige omrøringstankreaktorer, reaktorer sammenkoblet i serier, pluggjennomstrømningsreaktorer, pakkede kolonner eller tårn, eller andre kontinuerlige gjennomstrømninger som er klart innenfor området til en med kunnskap på feltet. The method with regard to the invention can be carried out continuously. E.g. contacting a fluid with the bacterial culture can be done using continuous stirred tank reactors, reactors connected in series, plug flow reactors, packed columns or towers, or other continuous flows that are readily within the skill of the art.
Stamme CVO er en Gram-negativ stav, 0,4 um i diamter og 0,5 til 2,0 (im i lengde, ikke-bevegelig under standard kulturbetingelser og ikke sporedannende. Den vokser anaerobt med ingen vekst observert under mikroaerofile betingelser. Stamme FWKO B er en Gram-negativ stav, 0,4 um i diameter og 2,0-4,0 um i lengde, bevegelig og ikke-sporedannende. Denne stamme er muligens mikroaerofil (pga. vekst i gradientmedia med sulfid og oksygen) og vokser like godt anaerobt. Disse to stammer CVO (NRRL B-21472) og FWKO B (NRRL B-21473) er blitt videre kjennetegnet som følger i tabell II. Tester for spesifisitet av 16S rRNA målrettede prober ble utført ved å bruke hele celler. Celler ble plassert i flekker på en nylonmembran i en konsentrasjon på 5 x 10<7>/spalte og lysert ved oppvarming (baking) i henhold til fremgangsmåten til Braun-Howland et al. (Braun-Howland, E.B., Vescio, P.A., og Nierzwici-Bauer, S.A., 1993, Use of a simplified Cell Blot Technique and 16S rRNA-Directed Probes for Identification of Common Environmental Isolates, Appl. Environ. Microbiol., 59:3219-3224). Blottene ble forvasket to ganger med 1 x SET-buffer (0,15 M NaCl, 1 mM EDTA, 0,02 M Tris; avsluttende pH-verdi 7,8) som inneholder 0,1% SDS, hybridisert over natten med radiomerket oligonukleotidprobe, vasket flere ganger med SET-buffer som inneholder 0,1 % SDS, og visualisert ved autoradiografi. Celler fra nært beslektede slekter (Thiobacillus denitrificans, Thiomicrospira denitrificans, Sulfurospirillum deleyianum, Arcobacter nitrofigilis, Campylobacter sp. DSM806), såvel som andre saltoppløsningsisolater ble brukt som negative kontroller. I tillegg ble blottene probet med en generell eubakteriell probe (EUB) som en positiv kontroll (se Braun-Howland et al; ovenfor). Strain CVO is a Gram-negative rod, 0.4 µm in diameter and 0.5 to 2.0 (µm in length, non-motile under standard culture conditions and non-spore-forming. It grows anaerobically with no growth observed under microaerophilic conditions. Strain FWKO B is a Gram-negative rod, 0.4 µm in diameter and 2.0-4.0 µm in length, motile and non-spore-forming. This strain is possibly microaerophilic (due to growth in gradient media with sulfide and oxygen) and grow equally well anaerobically. These two strains CVO (NRRL B-21472) and FWKO B (NRRL B-21473) have been further characterized as follows in Table II. Tests for specificity of 16S rRNA targeting probes were performed using whole cells. Cells were spotted onto a nylon membrane at a concentration of 5 x 10<7>/slot and lysed by heating (baking) according to the method of Braun-Howland et al (Braun-Howland, E.B., Vescio, P.A., and Nierzwici-Bauer, S.A., 1993, Use of a simplified Cell Blot Technique and 16S rRNA-Directed Probes for Identification of Common Environmental Isolates, Appl. Environment. Microbiol., 59:3219-3224). Blots were prewashed twice with 1x SET buffer (0.15 M NaCl, 1 mM EDTA, 0.02 M Tris; final pH 7.8) containing 0.1% SDS, hybridized overnight with radiolabeled oligonucleotide probe , washed several times with SET buffer containing 0.1% SDS, and visualized by autoradiography. Cells from closely related genera (Thiobacillus denitrificans, Thiomicrospira denitrificans, Sulfurospirillum deleyianum, Arcobacter nitrofigilis, Campylobacter sp. DSM806), as well as other saline isolates were used as negative controls. In addition, the blots were probed with a general eubacterial probe (EUB) as a positive control (see Braun-Howland et al; above).
En av probene testet reagerte spesifikt med stamme CVO og celler oppnådd fra en anrikning av produksjonssaltoppløsning (designert 59-20). Spesifisiteten til proben ble demonstrert ved mangel på hybridisering til andre lignende arter og isolater. Hybridisering av proben til cellene fra produksjonssaltoppløsningen anga nærvær av lignende bakterier i denne prøve. Den generelle eubakterielle probe, EUB, reagerte med alle prøvene som ventet. One of the probes tested reacted specifically with strain CVO and cells obtained from an enrichment of production saline (designated 59-20). The specificity of the probe was demonstrated by the lack of hybridization to other similar species and isolates. Hybridization of the probe to the cells from the production saline indicated the presence of similar bacteria in this sample. The general eubacterial probe, EUB, reacted with all samples as expected.
Den andre Campylobacter-lignende art, designert FWKO B (NRRL B-21473), som var lik men forskjellig fra stamme CVO som bestemt ved kromosomale hybridiseringsstudier ble også isolert og renset. The second Campylobacter-like species, designated FWKO B (NRRL B-21473), which was similar to but different from strain CVO as determined by chromosomal hybridization studies was also isolated and purified.
På basis av informasjonen diskutert og demonstrert ovenfor er begge stammene CVO og FWKO B ment å være stammer av Campylobacter species, og er referert til som Campylobacter-lignende arter i denne søknad. Based on the information discussed and demonstrated above, both strains CVO and FWKO B are believed to be strains of Campylobacter species, and are referred to as Campylobacter-like species in this application.
Med hensyn til en andre utforming av foreliggende oppfinnelse, en fremgangsmåte som kan anvendes i applikasjoner såsom oksidering av et sulfid i en fluid såsom saltoppløsning, olje eller gass er tilveiebragt. Fremgangsmåten omfatter eller består essensielt av, eller består av, å bringe en fluid i kontakt med en bakteriekultur som omfatter eller består essensielt av, eller består av, en bakterie som er istand til å oksidere sulfid, som er en Campylobacter species. Området og andre beskrivelser av bakteriekulturen og fluidet er det samme som de beskrevet i den første utforming av oppfinnelsen. With respect to a second embodiment of the present invention, a method which can be used in applications such as oxidation of a sulphide in a fluid such as salt solution, oil or gas is provided. The method comprises or consists essentially of, or consists of, bringing a fluid into contact with a bacterial culture which comprises or consists essentially of, or consists of, a bacterium capable of oxidizing sulphide, which is a Campylobacter species. The area and other descriptions of the bacterial culture and the fluid are the same as those described in the first design of the invention.
Å bringe fluidet i kontakt med bakteriekulturen kan utføres med hvilket som helst middel kjent for den med kunnskap på området. F.eks. kan bakteriekulturen som inneholder de nødvendige veks tel emen ter tilsettes til en fluid i en tilstrekkelig tidsperiode til hovedsakelig å redusere en sulfidforbindelse. Deretter kan bakteriekulturen eller brukt vekstmedium adskilles fra fluidet. Fluidet som har redusert fluidinnhold kan deretter anvendes i et utvalg av applikasjoner. Fordi vekst av en bakterie og adskillelse av en fluid fra bakteriell cellemasse og brukt. vekstmedium er vel kjent for en med kunnskap på området, er denne beskrivelse unngått her for korthets skyld. Bringing the fluid into contact with the bacterial culture can be accomplished by any means known to one skilled in the art. E.g. the bacterial culture containing the necessary metabolic elements can be added to a fluid for a sufficient period of time to substantially reduce a sulfide compound. The bacterial culture or used growth medium can then be separated from the fluid. The fluid which has a reduced fluid content can then be used in a selection of applications. Because growth of a bacterium and separation of a fluid from bacterial cell mass and used. growth medium is well known to someone with knowledge in the area, this description is avoided here for the sake of brevity.
I noen anvendelser, såsom f.eks. økt oljegjenvinning som involverer injeksjon'av en fluid såsom en saltoppløsning i en undergrunnsformasjon, behøver ikke bakteriekulturen og brukt medium å separeres fra fluidet. Bakteriekulturen i en saltoppløsning kan injiseres i en formasjon. Formasjonstypen er generelt ikke viktig og injeksjonen kan utføres ved ethvert middel kjent for den med kunnskap på området, såsom f.eks. pumping. In some applications, such as e.g. enhanced oil recovery involving the injection of a fluid such as a brine into a subsurface formation, the bacterial culture and spent medium do not need to be separated from the fluid. The bacterial culture in a saline solution can be injected into a formation. The type of formation is generally not important and the injection can be carried out by any means known to one skilled in the art, such as e.g. pumping.
Alternativt, kan en fluid såsom sulfidholdig gass tilsettes til en bakteriekultur som inneholder vekstmediet. Tilsetting av gassfluid til et vandig medium kan utføres ved ethvert middel kjent for en med kunnskap på området såsom f.eks. å boble gassfluidet inn eller gjennom det vandige medium. Alternatively, a fluid such as sulphide-containing gas can be added to a bacterial culture containing the growth medium. Addition of gas fluid to an aqueous medium can be carried out by any means known to one skilled in the art such as e.g. to bubble the gas fluid into or through the aqueous medium.
Tiden nødvendig for kontakten mellom et fluid med en bakteriell kultur beskrevet i en andre utforming av foreliggende oppfinnelse kan være ethvert tidsavsnitt så lenge som det er tilstrekkelig til å utvirke oksidasjon av et sulfid i fluidet. Nødvendig tid kan også være avhengig av konsentrasjoner av både sulfid og bakterielle celler i fluidet og kan være så kort som ca. 30 minutter til så lenge som ca. 1 uke. F.eks., hvis konsentrasjonen av inokulum er IO<7> celler/ml og sulfidkonsentrasjonen i fluidet er ca. 5 mM, kan det ta ca. 2 til ca. 20 timer å hovedsakelig oksidere sulfidet. The time necessary for the contact between a fluid with a bacterial culture described in a second embodiment of the present invention can be any period of time as long as it is sufficient to effect oxidation of a sulphide in the fluid. The time required may also depend on concentrations of both sulphide and bacterial cells in the fluid and may be as short as approx. 30 minutes to as long as approx. 1 week. For example, if the concentration of inoculum is 10<7> cells/ml and the sulphide concentration in the fluid is approx. 5 mM, it may take approx. 2 to approx. 20 hours to mainly oxidize the sulphide.
De følgende eksempler er tilveiebragt for å illustrere foreliggende oppfinnelse og er ikke ment uten grunn å begrense området for foreliggende oppfinnelse. Veksttemperaturen var der hvor ikke annet er angitt 30°C. The following examples are provided to illustrate the present invention and are not intended to unreasonably limit the scope of the present invention. The growth temperature was, where not otherwise stated, 30°C.
EKSEMPEL I EXAMPLE I
Dette eksempel demonstrerer biologisk mediert sulfidoksidasjon ved å bruke anrikede kulturer av saltoppløsning oppsamlet ved Coleville Unit, Saskatchewan. This example demonstrates biologically mediated sulfide oxidation using enriched cultures of brine collected at the Coleville Unit, Saskatchewan.
Oljereservoirsaltoppløsning med lav salinitet (0,71% totalt oppløste faststoffer) ble oppsamlet i sterile flasker under strengt anaerobe betingelser, fra en sandstensformasjon i Saskatchewan av Phillips Petroleum Company. Salt-oppløsningen ble oppsamlet fra en olje/vann separatortank på et punkt som var før reinjeksjon inn i i reservoiret (heretter referert til som injeksjonssaltoppløsning). Preparering av alle media og kulturer inkludert inkuberinger ble utført under anaerobe betingelser. De viktigste ioner tilstede i saltoppløsningen var natrium (0,29%), klorid (0,41%), bikarbonat (0,19%), sulfat (0,026%) og ammoniakk (0,001%) og pH-verdien var 7,5. Saltoppløsningen inneholdt 3,3 mM oppløselig sulfid som ble antatt å være dannet biologisk pga. den moderate reservoirtemperatur (30-35°C). Sulfid ble bestemt kolometrisk ved å bruke en metylenblåttmetode. Se også Fogo, L.K. og Popowski, M.; Spectrophotometric Determination of Hydrogen Sulfide, Anal. Biochem. 21:732-734 (1949). Fordi fremgangsmåten er vel kjent for en med kunnskap på området, er beskrivelsen utelatt heri. Low salinity oil reservoir brine (0.71% total dissolved solids) was collected in sterile bottles under strictly anaerobic conditions, from a sandstone formation in Saskatchewan by the Phillips Petroleum Company. The salt solution was collected from an oil/water separator tank at a point prior to reinjection into the reservoir (hereafter referred to as injection salt solution). Preparation of all media and cultures including incubations were performed under anaerobic conditions. The most important ions present in the salt solution were sodium (0.29%), chloride (0.41%), bicarbonate (0.19%), sulfate (0.026%) and ammonia (0.001%) and the pH value was 7.5 . The salt solution contained 3.3 mM soluble sulphide which was assumed to have been formed biologically due to the moderate reservoir temperature (30-35°C). Sulfide was determined colorimetrically using a methylene blue method. See also Fogo, L.K. and Popowski, M.; Spectrophotometric Determination of Hydrogen Sulfide, Anal. Biochem. 21:732-734 (1949). Because the method is well known to one skilled in the art, the description is omitted here.
Det totale antall bakterier tilstede ble anslått til 0,5-1,0 x 10<7> celler/ml ved direkte telling under anvendelse av akridinorange. Fermenterende, denitrifiserende, sulfatreduserende og sporedannende bakterier var alle tilstede i saltoppløsningen som vist ved vekst på anrikingskulturer og på agarplater. SRB representerte tilnærmet 1% av den mikrobielle populasjon på dette sted eller 10<4->10<5>/ml ved å bruke et laktatmedium formulert av American Petroleum Institute (API). Assayene ble satt opp i triplikat som brukt i "mest sannsynlige tall" (MPN)-assay. Anslag av tall ble for enkelthets skyld imidlertid gjort fra rådata heller enn å utføre en MPN-beregning. The total number of bacteria present was estimated to be 0.5-1.0 x 10<7> cells/ml by direct counting using acridine orange. Fermenting, denitrifying, sulfate-reducing, and spore-forming bacteria were all present in the saline as shown by growth on enrichment cultures and on agar plates. SRB represented approximately 1% of the microbial population at this site or 10<4->10<5>/ml using a lactate medium formulated by the American Petroleum Institute (API). The assays were set up in triplicate as used in the "most probable number" (MPN) assay. For the sake of simplicity, estimates of numbers were, however, made from raw data rather than carrying out an MPN calculation.
Det ble funnet at sulfidoksidasjon fant lett sted når nitrat og fosfat ble satt til saltoppløsningsanrikingskulturer. F.eks. etter tilsetting av 5 mM KNO3 og 100 uM KH2PO4, ble sulfidnivået redusert fra 3,3 mM til et ikke-detekterbart nivå (< 0,1 mM) på 48 timer ved 30°C. I kontrast var det ingen forandring i kontrollmediet som ikke inneholdt nitratet. Med tilsatt fosfat og nitrat var det en 10-ganger økning i celletall ved direkte telling sammenlignet med kontroller, noe som anga at vekst foregikk. Lignende hastigheter for sulfidoksidasjon ved å bruke saltoppløsninger fra tre produksjonsbrønner ble også observert og antyder at sulfidoksiderende bakterier er fordelt gjennom formasjonen. It was found that sulfide oxidation occurred readily when nitrate and phosphate were added to saline enrichment cultures. E.g. after addition of 5 mM KNO3 and 100 uM KH2PO4, the sulfide level was reduced from 3.3 mM to a non-detectable level (< 0.1 mM) in 48 h at 30°C. In contrast, there was no change in the control medium that did not contain the nitrate. With phosphate and nitrate added, there was a 10-fold increase in cell numbers by direct count compared to controls, indicating that growth was occurring. Similar rates of sulfide oxidation using brines from three production wells were also observed and suggest that sulfide oxidizing bacteria are distributed throughout the formation.
Det ble også funnet at økende nivåer av nitratstimulért sulfidoksidasjon, opptil 2,5 mM, på hvilket punkt sulfidnivået ble redusert til ikke-detekterbart. Disse resultatene demonstrerer at sulfidoksidasjon var nitratavhengig. It was also found that increasing levels of nitrate stimulated sulfide oxidation, up to 2.5 mM, at which point sulfide levels were reduced to undetectable. These results demonstrate that sulfide oxidation was nitrate dependent.
Analyser av brukte anrikingskulturer beskrevet ovenfor for sulfat, sulfitt og total oppløselig svovel anga at et oppløselig svoveloksidasjonsprodukt såsom sulfat ikke ble akkumulert. Under oksidasjonsprosessen kom imidlertid et gulhvitt bunnfall tilsyne i anrikingsflaskene. Analyse av dette uoppløselige materiale ved røntgendifraksjon og elektrondispersjonspektroskopi anga at det var en blanding av element ert svovel og kalsitt. Nitratreduksjonen resultert i dannelse av nitrit og nitrogengass. Det var ingen netto økning i ammoniakk. Analyzes of spent enrichment cultures described above for sulfate, sulfite, and total soluble sulfur indicated that a soluble sulfur oxidation product such as sulfate was not accumulated. During the oxidation process, however, a yellowish-white precipitate became visible in the enrichment bottles. Analysis of this insoluble material by X-ray diffraction and electron dispersion spectroscopy indicated that it was a mixture of elemental sulfur and calcite. Nitrate reduction resulted in the formation of nitrite and nitrogen gas. There was no net increase in ammonia.
EKSEMPEL II EXAMPLE II
Dette eksempel illustrerer opptelling og identifikasjon av sulfidoksiderende bakterier. Dette eksempel demonstrerer også oksidasjon av sulfid i saltopp-løsninger og syntetiske media ved å bruke bakteriene i henhold til foreliggende oppfinnelse. This example illustrates enumeration and identification of sulfide-oxidizing bacteria. This example also demonstrates the oxidation of sulfide in saline solutions and synthetic media using the bacteria of the present invention.
Nitratreduserende sulfidoksiderende bakterier ble opptelt med MPN, som beskrevet 1 eksempel I, ved å bruke oksidasjon av redoksindikatoren resazurin som en vekstindikator. Bruk av indikatoren resazurin er en fremgangsmåte som er vel kjent for en med kunnskap på området. Tilnærmet 10<4> sulfatoksiderende bakterier/ml var tilstede i prøvene fra injeksjonssaltoppløsningen og prøvene fra tre produserende brønner. Plating av de anrikede kulturer fra injeksjonssaltoppløsning på 295 agar medium (se fotnote, Tabell II) resulterte i rensing av flere kolonityper av bakterier. En av de oppnådde isolatene, CVO, (NRRL B-21472), var en Gram-negativ stav, istand til å oksidere sulfid når den ble inokulert i filtersterilisert saltoppløsning supplementert med nitrat og fosfat (se fig. 1). Filtersterilisert saltoppløsning var Coleville saltoppløsning oppsamlet ved fritt-vann-utskylling og filtrert gjennom et 0,2 um celluloseacetatfilter for å fjerne bakterielle celler. Inokulasjon ble gjort med 2 ml av en kultur som inneholdt ca. 10<7> celler pr. ml. Nitrate-reducing sulfide-oxidizing bacteria were enumerated by MPN, as described in Example I, using oxidation of the redox indicator resazurin as a growth indicator. Use of the indicator resazurin is a method that is well known to someone with knowledge in the field. Approximately 10<4> sulfate-oxidizing bacteria/ml were present in the samples from the injection saline solution and the samples from three producing wells. Plating of the enriched cultures from injection salt solution on 295 agar medium (see footnote, Table II) resulted in the purification of several colony types of bacteria. One of the isolates obtained, CVO, (NRRL B-21472), was a Gram-negative rod capable of oxidizing sulfide when inoculated in filter-sterilized saline supplemented with nitrate and phosphate (see Fig. 1). Filter-sterilized saline was Coleville saline collected by free-water rinsing and filtered through a 0.2 µm cellulose acetate filter to remove bacterial cells. Inoculation was done with 2 ml of a culture containing approx. 10<7> cells per ml.
Fig. 1 viser oksidasjon av sulfid av stamme CVO i filtrert saltoppløsning supplementert med 5 mM KNO3 og 100 uM natriumfosfat (NaH2P04). Uten nærvær av CVO-celler (-CVO, fig. 1), var det liten eller ingen sulfidoksidasjon. I nærvær av cellene av stamme CVO (+CVO, fig. 1), skjedde sulfidoksidasjon imidlertid hurtig. Lignende resultater ble oppnådd når syntetisk medium CSB/DTA ble brukt istedenfor filtrert saltoppløsning (fig. 2). Med i esamm en setningen til CSB/DTA er vist i tabell III. Resultatene vist i fig. I og fig. 2 viser at bakteriene i henhold til foreliggende oppfinnelse katalyserte oksidasjonen av sulfid inn i enten oljefeltsaltoppløsninger eller i syntetiske media. Fig. 1 shows oxidation of sulfide by strain CVO in filtered saline supplemented with 5 mM KNO3 and 100 uM sodium phosphate (NaH2PO4). In the absence of CVO cells (-CVO, Fig. 1), there was little or no sulfide oxidation. However, in the presence of the cells of strain CVO (+CVO, Fig. 1), sulfide oxidation occurred rapidly. Similar results were obtained when synthetic medium CSB/DTA was used instead of filtered saline (Fig. 2). Together, the sentence for CSB/DTA is shown in table III. The results shown in fig. I and fig. 2 shows that the bacteria according to the present invention catalyzed the oxidation of sulphide into either oilfield salt solutions or into synthetic media.
Fig. 3 illustrerer økning av sulfidoksidasjon ved å tilsette CVO-celler til naturlig produsert saltoppløsning. Kjøringen ble utført som følger. Anrikinger med en ufiltrert produksjon av saltoppløsning som har sammensetningen som vist i CSB basis (første fem linjer i tabell III), som inneholder 4,4 mM oppløselig sulfid ble fremstilt ved å tilsette 50 ml saltoppløsning, 5 mM KN03 og 100 uM NaH2P04 til serumflasker. I et tilfelle ble saltoppløsningen supplementert med 2 ml (ca. 10 celler/ml) av en kultur av stamme CVO som var dyrket natten over. Tilsetting av stammen CVO reduserte forsinkelsestiden betydelig og reduserte den tid som var nødvendig for fullstendig oksidasjon av sulfidet fra mer enn ca. 34 timer til mindre enn ca. 12 timer. Fig. 3 illustrates the increase of sulphide oxidation by adding CVO cells to naturally produced salt solution. The driving was carried out as follows. Enrichments with an unfiltered production saline having the composition shown in the CSB basis (first five lines of Table III), containing 4.4 mM soluble sulfide were prepared by adding 50 ml of saline, 5 mM KN0 3 and 100 uM NaH 2 PO 4 to serum bottles . In one case, the saline solution was supplemented with 2 ml (about 10 cells/ml) of a culture of strain CVO grown overnight. Addition of the strain CVO significantly reduced the lag time and reduced the time required for complete oxidation of the sulfide from more than ca. 34 hours to less than approx. 12 hours.
EKSEMPEL III EXAMPLE III
Dette eksempel illustrerer sulfidoksidasjonshastighet til Campylobacter-lignende arter i henhold til foreliggende oppfinnelse. This example illustrates the sulfide oxidation rate of Campylobacter-like species according to the present invention.
Kjøringene ble utført med fritt-vanns væskeutskillingstanker (FWKO) saltopp-løsning som beskrevet i eksempel II. Saltoppløsningen, satt opp i triplikat, ble filtersterilisert og supplementert med 5 mM KNO3 og 100 uM NaH2P04 (stamløsning av disse ble sterilisert separat). Hver kjøring vist i tabell IV nedenfor ble inokulert med kulturer som inneholder ca. 10<7> celler/ml som notert i tabell IV. Sulfid ble målt som beskrevet i eksempel I. The runs were carried out with free-water liquid separation tanks (FWKO) saline solution as described in example II. The salt solution, set up in triplicate, was filter sterilized and supplemented with 5 mM KNO3 and 100 uM NaH2PO4 (stock solution of these was sterilized separately). Each run shown in Table IV below was inoculated with cultures containing approx. 10<7> cells/ml as noted in Table IV. Sulfide was measured as described in Example I.
Resultatene vist i tabell IV angir at sulfidnivåene i filtrert saltoppløsningskontroll ble hovedsakelig ikke forandret i løpet av 14 timer. Resultatene viser også at hastigheter og forsinkelsestid for sulfidoksidasjon var lik for begge stammer av CVO og FWKO B og ufiltrert saltoppløsningsanrikelse (siste kolonne, tabell IV). The results shown in Table IV indicate that sulfide levels in the filtered saline control were essentially unchanged over 14 hours. The results also show that rates and lag time of sulphide oxidation were similar for both strains of CVO and FWKO B and unfiltered saline enrichment (last column, Table IV).
Etter 24 timers inkubasjon ble celletellinger for kulturene til disse fire kjøringene vist i tabell IV bestemt ved direkte mikroskopisk telling og bruk av akridin orange. Resultatene vist i tabell IV ovenfor angir at det endelige celleantall, unntagen for den filtrerte saltoppløsningskontroll, var tilnærmet de samme. After 24 hours of incubation, cell counts for the cultures of these four runs shown in Table IV were determined by direct microscopic counting and the use of acridine orange. The results shown in Table IV above indicate that the final cell counts, except for the filtered saline control, were approximately the same.
Resultatene vist i de ovenstående eksempler demonstrerer klart at den foreliggende oppfinnelse er godt tilpasset til å utføre de hensikter og nå de mål og har de fordeler som er nevnt, såvel som de som ligger innebygget deri. The results shown in the above examples clearly demonstrate that the present invention is well adapted to carry out the purposes and achieve the goals and has the advantages mentioned, as well as those inherent therein.
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US08/499,721 US5686293A (en) | 1995-07-07 | 1995-07-07 | Sulfide-oxidizing bacteria |
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US5989513A (en) * | 1995-07-28 | 1999-11-23 | Gas Research Institute | Biologically assisted process for treating sour gas at high pH |
EP0845288A1 (en) * | 1996-11-27 | 1998-06-03 | Thiopaq Sulfur Systems B.V. | Process for biological removal of sulphide |
US20030201227A1 (en) * | 1996-12-17 | 2003-10-30 | Perriello Felix Anthony | Remediation of odorous media |
US7182871B2 (en) * | 1996-12-17 | 2007-02-27 | Global Biosciences, Inc. | Wastewater treatment with alkanes |
US7192523B2 (en) * | 1996-12-17 | 2007-03-20 | Global Biosciences, Inc. | Methods for treating agricultural waste and producing plant growth-enhancing material |
US6669846B2 (en) * | 1996-12-17 | 2003-12-30 | Global Biosciences, Inc. | Wastewater treatment with alkanes |
US6835312B2 (en) * | 2001-05-15 | 2004-12-28 | Global Biosciences, Inc. | Method and apparatus for treatment of septic systems with alkane-utilizing bacteria |
US6306288B1 (en) | 1998-04-17 | 2001-10-23 | Uop Llc | Process for removing sulfur compounds from hydrocarbon streams |
US7329532B2 (en) * | 2001-07-27 | 2008-02-12 | Global Biosciences, Inc. | Remediation of sulfur-containing pollutants with hydrocarbon-utilizing bacteria |
US20030185296A1 (en) * | 2002-03-28 | 2003-10-02 | Masten James W. | System for the capture of evidentiary multimedia data, live/delayed off-load to secure archival storage and managed streaming distribution |
US7833551B2 (en) | 2004-04-26 | 2010-11-16 | Conocophillips Company | Inhibition of biogenic sulfide production via biocide and metabolic inhibitor combination |
US7101410B1 (en) | 2004-06-03 | 2006-09-05 | Baugh Clarence L | Method for the microbiological desulfurization of fossil fuels |
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US20080190844A1 (en) * | 2007-02-13 | 2008-08-14 | Richard Alan Haase | Methods, processes and apparatus for biological purification of a gas, liquid or solid; and hydrocarbon fuel from said processes |
US7998724B2 (en) * | 2007-04-27 | 2011-08-16 | Ut-Battelle Llc | Removal of mercury from coal via a microbial pretreatment process |
US20090005601A1 (en) * | 2007-06-29 | 2009-01-01 | Gaylord Chemical Company Llc | Process for preparing low malodor dimethyl sulfoxide |
US8573300B2 (en) * | 2011-09-07 | 2013-11-05 | E I Du Pont De Nemours And Company | Reducing sulfide in oil reservoir production fluids |
US20130056214A1 (en) * | 2011-09-07 | 2013-03-07 | E. I. Du Pont De Nemours And Company | Reducing sulfide in production fluids during oil recovery |
US9739132B2 (en) * | 2013-08-07 | 2017-08-22 | Baker Hughes Incorporated | Well treatment fluids and methods |
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US5236677A (en) * | 1992-03-13 | 1993-08-17 | Grupo Cydsa S.A. De C.V. | Biological process for the elimination of sulphur compounds present in gas mixtures |
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